We are studying the mechanism whereby viruses enter their host cells and initiate replication. The virus systems to be studied are Semliki Forest virus (SFV) and Influenza A virus, which have proven relevant model systems for the general problems of early virus-cell interactions. The overall aim is to define on molecular and cellular terms the following steps in entry: internalization of the virus particles by endocytosis, penetration from endosomes by membrane fusion, uncoating of the genome, and transport to the cytoplasmic or nuclear location of replication and transcription. We hope to obtain a deeper understanding of the principles underlying viral pathogenicity and cell tropism as well as of basic biological phenomena such as membrane fusion, nuclear targeting and the interaction between cellular compartments. The specific aspects include a more detailed definition of endocytic organelles where acid-triggered fusion of the viruses takes place. The conformational changes in the viral fusion proteins acid-induced, activity will be characterized in detail with emphasis on the sterol dependence of SFV (now recognized as a common property among viruses) and the interaction between the fusion factors and the fusing membranes. The mechanism of action of the trimeric hemagglutinin, the fusion factor of Influenza virus, will be studied using hybrid molecules made up of trimers consisting of different subunits. The fate of the nucleocapsids after penetration will be followed by biochemical and immunochemical methods. We will, moreover, try to resolve the signals which induce uncoating of the genome and analyze the molecular mechanism by which uncoating takes place, the transport of the genome to the SFV-specific cytopathic vacuols, and Influenza to the nucleus, will be investigated. A variety of methods ranging from immunocytochemistry on frozen sections to bulk delivery of anti-sense oligonucleotides will be used.